This invention relates to the fields of biochemistry and medicine, and in particular to novel liposomal formulations and process for making such formulations. More specifically, this invention relates to liposomal formulations containing camptothecin and analogs thereof. Further, this invention relates to methods of manufacturing and of using such formulations.
Camptothecin is a pentacyclic plant alkaloid originally isolated from the bark of Camptotheca accuminata trees indigenous to China (Wall et al., J. Am. Chem. Soc., 94: 388 (1966)). The drug contains a fused ring structure incorporating quinoline, pyrrolidine, alpha-pyridone and a six membered lactone ring. The naturally occurring form of camptothecin is optically active, with the asymmetric carbon atom at position 20 of the lactone ring in the xe2x80x9cSxe2x80x9d configuration. Camptothecin and numerous analogs thereof (hereafter termed camptothecins) are currently the focus of intensive study due to the potent anti-tumor activity displayed by these compounds both in vitro and in vivo (e.g., Giovanella, et al, Science 246: 1046-1048 (1989)). The cytotoxic effects of camptothecins have also been exploited in their use as anti-viral, anti-Plasmodium and anti-haemoflagellate agents (Priel et al., U.S. Pat. No. 5,622,959; Priel et al., U.S. Pat. No. 5,422,344; Atlas, WO 9611005; Wall et al., U.S. Pat. No. 5,614,529; Shapiro et al., U.S. Pat. No. 5,496,830; Pardee, WO 9404160).
Camptothecin drugs are believed to exert their anti-tumor effect by binding to and reversibly inhibiting the action of the enzyme topoisomerase I. This enzyme is required for DNA and RNA synthesis in proliferating cells, where it catalyses the relaxation of supercoiled DNA structures that form during these processes. As a result of topoisomerase I inhibition, the biosynthesis of nucleic acids is strongly inhibited leading to DNA breakage and cell death.
Despite their impressive antitumor properties, the use of camptothecins includes a number of drawbacks. First, some camptothecins are extremely insoluble in aqueous solution, making parenteral administration of the drug problematic. Second, the lactone ring is susceptible to hydrolysis at the pH of blood plasma, resulting in a carboxylate form of the drug that has significantly reduced topoisomerase activity (Fassberg and Stella (1992) J. Pharm. Sci. 81(7):676-689; Mi et al. (1995) Biochemistry 34(42):13722-13728; Potmesil (1994) Cancer Res. 54:1431-1439; Slichenmyer et al. (1993) J. Natl. Cancer Inst. 85:271-291; Jaxel et al. (1989) Cancer Res. 49:5077-5082). Moreover, the carboxylate form is highly toxic, often inducing gastrointestinal toxicity, myelosuppression and hemorrhagic cystitis. The biologically active lactone form is also toxic.
As a result of these limitations, much effort has been invested in the synthesis of semi-synthetic camptothecin derivatives with increased water solubility, decreased toxicity and increased resistance to hydrolysis. Two examples are topotecan (Hycamtin(trademark)) (Kingsbury et al., J. Med. Chem. 34:98 (1991); Boehm et al., European Patent Application No. 321,122) which is approved for salvage therapy of metastatic ovarian carcinoma, and irinotecan (Miysaka et al., U.S. Pat. No. 4,604,463) which is approved for salvage therapy of colon cancer. Other derivatives of camptothecin and anti-tumor treatments using these derivatives are described in Wall et al. (U.S. Pat. No. 5,340,817); Wall et al. (U.S. Pat. No. 5,364,858); Wall et al. (U.S. Pat. No. 5,244,903); Wall (U.S. Pat. No. 5,180,722); Wall et al. (U.S. Pat. No. 5,227,380); Wall et al. (U.S. Pat. No. 5,049,668); Wani et al. (U.S. Pat. No. 5,122,606); Giovanella et al. (U.S. Pat. No. 5,225,404); Giovanella et al. (U.S. Pat. No. 5,552,154); Wall el al. (U.S. Pat. No. 5,401,707); Wall el al. (U.S. Pat. No. 5,122,526); Johnson et al. (WO 9311770); Johnson et al. (WO 9214471); Johnson et al. (WO 9214469); and Cherian (WO 9611669).
Recently, Luzzio et al. (U.S. Pat. No. 5,559,235) disclosed a camptothecin derivative that is moderately soluble in aqueous media, while retaining topoisomerase I activity in vitro and anti-tumor activity in vivo. This compound, 7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin dihydrochloride, (also known as GI147211C or lurtotecan) is less toxic overall than camptothecin, and is 5-10 times more potent than topotecan in tumor cell cytotoxicity assays (Emerson et al. (1995) Cancer Research 55:603-609). Some of the stability samples from the clinical trial formulations of GI147211C were reported to contain a sulfate-GI147211C precipitate as a result of residual levels of sulfate in glass vials (Tong et al. (1996) PDA Journal of Pharmaceutical Science and Technology 50(5):326-329). As a practical and efficient therapeutic it would be desirable to administer this compound in a manner that provides an improved therapeutic index.
Insoluble, hydrolyzable compounds may be administered in a clinical situation by packaging the compounds into lipid aggregates or constructs such as liposomes or micelles. Liposomes are known to be physiologically compatible and biodegradable delivery systems for a broad range of drugs. Furthermore, as solvation in aqueous media is not required, an insoluble compound can be delivered to the site of action in a more concentrated and easily administered medicament than the free drug alone.
Burke (U.S. Pat. No. 5,552,156) discloses liposome-associated camptothecins in which it is postulated that the lactone ring of the camptothecin intercalates with the acyl chains of the lipid bilayer. The lactone ring is effectively removed from the aqueous environment in the interior of the liposome, and protected from hydrolysis. Burke also describes how the pH of the internal liposome compartment can be lowered so that camptothecin derivatives with lower affinity for the liposome membranes can reside within the aqueous interior without undergoing hydrolysis. In vivo tests demonstrating the stability of the formulations were not performed.
Constantinides et al. (WO 95/08986) discloses formulations of camptothecin and its structurally related analogs in liposomes having at least 80% of the drug incorporated in the lipid bilayer. Pharmacokinetic studies were performed on an egg phosphatidylcholine, egg phosphatidylglycerol, camptothecin formulation. The liposomes were multilamellar and the camptothecin was passively entrapped. Increased plasma levels of camptothecin were observed over a 4-hour period after administration of the liposomal formulation as compared to those obtained with the free drug. A larger area under the plasma concentration-time curve (AUC) was obtained from liposomal versus free camptothecin, although the increase of the AUC was only about 4 fold higher.
Camptothecins incorporated within vesicles and liposomes are also described in Slater el al. (WO 9426253) and Castor et al. (WO 9615774).
The present invention provides for liposomal formulations of camptothecin and its structurally related analogs as well as methods for their preparation. The liposomes have improved pharmacokinetics, enhanced efficacy as anti-tumor agents, and provide an improved therapeutic index as compared to the free drug. The formulations include liposomes comprising at least one phospholipid and a camptothecin or analog thereof (referred to collectively herein as xe2x80x9ccamptothecinxe2x80x9d). In one embodiment, the formulations include liposomes comprised of cholesterol, a phosphatidylcholine, an excipient, wherein the excipient is sulfate or citrate, and a camptothecin, wherein a portion of the camptothecin may be precipitated in the aqueous interior of the liposomes in the presence of the excipient. The preferred camptothecin for use in this invention is GI147211. Further, the formulations described herein are stable upon storage.